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Mechanisms of Bcl-2 in Programmed Cell Death Laura Beth Hill St. Edward’s University
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Apoptosis Essential for normal embryonic development Natural and pathological Morphologic characteristics Regulated by proteins in Bcl-2 family
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The Apoptotic Process Cell receives death signal Mitochondrial membrane potential decreases Transport of cytochrome c through membrane into cytosol Cytochrome c binds to Apaf-1 Caspase activity initiated Cell degradation
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What is Bcl-2? Family of proteins that includes promoters and inhibitors Proto-oncogene Localized to outer mitochondrial membrane
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Characteristics Bcl-2 family members can homo- and heterodimerize Participate in selective pore formation Expressed at different rates during development
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FOCUS OF SEMINAR Possible Mechanisms Bcl-2 blocks release of cytochrome c from mitochondrial membrane (Yang, et al., 1997) Bcl-2 forms channels in lipid membranes (Schendel, et al., 1997)
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Cytochrome c Model Necessary for the initiation of apoptosis Found in the mitochondrial intermembrane space Localization suggests connection between Bcl-2 and cytochrome c
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HL-60 cells neo cellsbcl-2 cells Isolation Staurosporine Immunoblot analysis
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Yang’s Results Cytochrome c in neo cells showed cytosol increase, with corresponding decrease in mitochondria No significant change of cytochrome c in mitochondria or cytosol of Bcl-2 cells
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Yang’s Conclusion Bcl-2 prevents the release of cytochrome c Mechanism by which Bcl-2 blocks release unknown Structural similarity to bacterial toxins suggests pore-forming ability
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Channel Formation Model Bcl-2 can regulate Ca 2+ fluxes and protein transport 3D structure of Bcl-x L is similar to the pore-forming domains of DT and the bacterial colicins
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Cells expressing Bcl-2 Mutant Wild-type Purification Detection of single channels Detection of single channels
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Schendel’s Results Bcl-2 formed ion-conducting pores in a manner similar to that of bacterial toxins Bcl-2 mutant produced only non-specific Cl - efflux Bcl-2 in planar lipid bilayers formed discrete cation-selective channels Bcl-2 mutant did not form channels here
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Schendel’s Conclusions Biophysical evidence proves that Bcl-2 forms channels in membranes Channels reside in closed state What controls opening and closing? What does Bcl-2 transport? How do pro-apoptotic proteins oppose anti-apoptotic proteins?
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Practical Importance Adjustment of apoptotic threshold Gene therapy to control neuronal death Protection of developing nervous system against neurotoxins (e.g. EtOH)
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Acknowledgements St. Edward’s School of Natural Sciences faculty and staff
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